1. Field of the Invention
The present invention relates to a method for analyzing a substance that has been developed over a membrane in a solid phase by using a laser desorption ionization mass spectrometric method in various fields such as clinical, diagnostic, biochemical and molecular biological fields.
2. Description of the Background Art
In order to analyze a mass of molecules to be measured, a laser desorption ionization mass spectrometric method has been used in which a laser beam is applied to a sample placed on a sample plate attached to a mass spectrometer so that the sample is ionized and analyzed (see JP-A No. 10-40858). Upon placing the sample on the sample plate so as to be analyzed, there are two methods, that is, one method in which a matrix is used and the other method in which a matrix is not used.
A method in which the method using a matrix is combined with a time-of-flight mass spectrometer is referred to as MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometric method.
In the MALDI-TOF method, a measuring sample is dropped onto a metal sample plate together with a matrix solution, and after having been dried, this is subjected to a measuring process. In this case, the sample to be dropped needs to be closely crystallized in a fixed area.
Here, with respect to the measuring sample, a mass spectrometric method has been proposed in which after biomolecules have been separated through electrophoresis or the like, these are transferred onto a membrane in a solid phase, and the solid-phase sample is subjected to various reactions on the membrane by utilizing a trace-amount application technique using a piezoelectric element, and the resulting reaction products are utilized to carry out mass analysis (see International Publication No. WO98/47006).
In the case where the sample, adsorbed on a membrane, is subjected to a mass spectrometric analysis, in comparison with MALDI-TOF measurements directly carded out on the corresponding reaction product on the membrane, it is more preferable to carry out measurements on the sample that has extracted from the membrane, and transferred onto an MALDI-TOF-use sample plate, in order to obtain measured values with higher precision.
Here, in an attempt to provide a device which carries out a sequence of processes of extracting a sample from the membrane, transferring the sample onto a sample plate and forming an MALDI-TOF-use sample plate, two plates (stages) are required. In other words, one plate on which a membrane holding a separated biological sample is fixedly held so that the target molecule adsorption position is recognized so as to apply a reagent and the other sample plate on which the sample extracted from the membrane is placed so as to be introduced to an MALDI-TOF mass spectrometer are required. When these plates are controlled by the same device, two plates or stages need to be placed on the same plane in parallel with each other, causing a limitation in reducing the device size.
The above explanation has exemplified a case in which the sample for use in MALDI-TOF measurements is prepared. However, the same problem arises also in the case where the sample is prepared without using a matrix.
The object of the present invention is to achieve a process for transferring a sample adsorbed on a membrane to a sample plate for a laser desorption ionization mass spectrometric method by using a small-size device.
In order to achieve the above-mentioned objective, the present invention provides an area used for fixedly holding a membrane bearing a sample adsorbed thereon on a sheet of a sample plate in addition to an area on which a sample used for mass spectrometry is placed.
That is, the sample preparation method of the present invention is a method for preparing a sample to be analyzed on a sample plate for a laser desorption ionization mass spectrometric method which applies a laser beam onto a sample placed on a sample plate attached to a mass spectrometer so that the sample is ionized, and then analyzed, and one portion of areas on the sample plate surface is prepared as an ionization area used for ionizing the sample through irradiation with a laser beam, and another portion on the sample plate surface is prepared as a plane area to which a membrane bearing the sample adsorbed thereon is fixed, and in this method, after the membrane bearing the sample adsorbed thereon has been fixedly held on the above-mentioned plane area, the sample is extracted from the membrane, and the extracted sample is placed on the above-mentioned ionization area so as to prepare an ionization-use sample.
The sample plate of the present invention is a sample plate which is used in a laser desorption ionization mass spectrometer, and attached to a mass spectrometer, with a sample to be analyzed being placed on the surface thereof, so that the sample is ionized through irradiation with a laser beam, and is characterized in that an ionization area which is used for ionizing the sample through irradiation with a laser beam and a plane area to which a membrane bearing the sample adsorbed thereon is fixed are prepared.
As described above, the ionization area used for ionizing the sample and the plane area to which the membrane bearing the sample adsorbed thereon is fixed are installed on the same plate so that it becomes possible to reduce the stage area to be used in the device, and consequently to miniaturize the entire device.
One of the preferable methods for ionization of the sample is a matrix-assisted laser desorption ionization method. In this case, a sample to be placed on the ionization area of the sample plate is prepared by using a matrix.
In one of the preferable examples for the method by which the membrane bearing the sample adsorbed thereon is fixedly held on the plane area of the sample plate, a medium in which the sample is developed is superposed on the membrane, and after the sample has been transferred from the medium onto the membrane by applying a voltage between the medium and the membrane, the membrane is fixedly held to a state in which the membrane is electrically conducted to the sample plate.
In a preferable mode, the ionization area of the sample plate is arranged so that portions on which respective samples are placed are separated from other portion by borders so that the samples are placed in a locally distributed manner. With respect to the borders, for example, grooves each of which surrounds the corresponding sample portion are formed.
Examples of the sample to be adsorbed on the membrane include molecules of proteins, peptides, saccharides, lipids, nucleic acid molecules and the like or a mixture of these molecules that are separated through SDS (sodium dodecyl sulfate) polyacrylamide electrophoresis, two-dimensional electrophoresis in which isoelectric focusing electrophoresis and SDS polyacrylamide electrophoresis are combined, or other chromatography processes.
These samples may be modified by a proteolytic enzyme, a glycolytic enzyme, nuclease or a combination thereof. The sample, modified in such a manner, can be extracted from the membrane by using a solvent The sample thus extracted is dropped onto the ionization area of the sample plate, and placed thereon.
With respect to the material of a membrane to be used for solid-phase deposition of a sample, examples thereof include PVDF (polyvinylidene difluoride), nitrocellulose, nylon (registered trademark) or derivatives thereof.
The present invention eliminates the necessity of separately preparing the membrane-fixing-use plate and the mass-measuring-use plate, thereby making it possible to cut costs required for the analysis, to reduce the area occupied by the respective plates in the device, and consequently to miniaturize the device.
Moreover, the sample plate may of course be used as a simple general-use mass spectrometry sample plate, and may be applied to a method for directly carrying out a mass spectrometric analysis of a sample adsorbed on a membrane; therefore, it is possible to provide a method in which one sheet of plate can be applied to many kinds of analyses.